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Blood, Vol. 113, Issue 1, 233-243, January 1, 2009
Endothelial progenitor cell homing: prominent role of the IGF2-IGF2R-PLCβ2 axis
Blood Maeng et al.
113: 233
Supplemental materials for: Maeng et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 68.6 KB)
- Table S1. Sequence of RT-PCR primers (PDF, 54.5 KB)
- Table S2. Sequence of Real time-PCR primers (PDF, 45.7 KB)
- Figure S1. Characterization of EPCs and OECs (JPG, 115 KB)
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(A) EPCs were isolated from human umbilical cord blood and cultured. After 7 d culture, EPC identification and estimation of culture purity (90–95%) were determined by the percentage of cells that are stained with FITC-labeled Ulex europaeus lectin I (UEA-1) and uptake DiI-conjugated acLDL (acetylated low-density lipoprotein) (B) EPCs were further cultured in endothelial basal medium-2 (EBM-2) supplemented with endothelial growth medium 2 (EGM-2) containing fetal bovine serum, human VEGF-A, human fibroblast growth factor, human epidermal growth factor, IGF1, and ascorbic acid in appropriate amounts and normally differentiated into OECs within 7–14 d. (C) Total mRNAs were isolated from EPCs and OECs and the marker gene expression profile was assessed by RT-PCR. (D), (E) and (F) EPCs and OECs were further characterized by flow cytometry and immunofluorescent staining.
- Figure S2. The effects of IGF1 and IGF2 on the chemotaxis of EPCs and OECs (JPG, 156 KB)
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(A) EPCs were treated with IGF1 (1, 10, and 100 ng/ml) for 24 h. (B) OECs were treated with IGF2 (1, 10, 50, and 100 ng/ml) or VEGF (50 ng/ml) for 4 h. (C) EPCs were preincubated for 30 min with or without IGF1RN (1, 10, and 100 µg/ml) and stimulated with IGF2 (50 ng/ml) for 24 h. Chemotaxis was quantified by counting the cells that migrated to the lower side of the filter with optical microscopy at 200× magnification.
- Figure S3. IGF2 induces CD34+ and CD34− EPCs through an IGF2R-mediated pathway (JPG, 40.1 KB)
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(A) CD34+ and CD34− EPCs were separated using the MACS system and IGF2R gene expression was assessed by RT-PCR. (B) and (C) CD34+ and CD34− EPCs were preincubated for 30 min with or without 5 mM M6P and stimulated with 50 ng/ml IGF2. After 24 h, chemotaxis was quantified by counting the cells that migrated to the lower side of the filter with optical microscopy at 200× magnification. Data are the mean ± S.E. **, p < 0.01 vs. IGF2 alone.
- Figure S4. IGF2 induces chemotaxis of mBMMNCs (JPG, 60 KB)
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EGFP CD34+ mBMMNCs were preincubated for 30 min with or without 5 mM M6P and stimulated with 50 ng/ml mouse IGF2 or 50 ng/ml mouse VEGF. After 24 h, chemotaxis was quantified by counting the cells that migrated to the lower side of the filter with fluorescence microscopy at 200× magnification.
- Figure S5. Hypoxia upregulates VEGF expression (JPG, 80.8 KB)
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(A) MDA-MB-435 breast cancer cells and HUVECs were incubated for the indicated times under either normoxic (N) or hypoxic (H) conditions. VEGF mRNA was measured by RT-PCR. (B) EPCs and OECs were characterized by RT-PCR, flow cytometry and immunofluorescent staining.
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